Focal Adhesion Kinase Activates Stat1 in Integrin-mediated Cell Migration and Adhesion
2001; Elsevier BV; Volume: 276; Issue: 22 Linguagem: Inglês
10.1074/jbc.m009063200
ISSN1083-351X
AutoresBing Xie, Jihe Zhao, Motoo Kitagawa, Joan E. Durbin, Joseph A. Madri, Jun‐Lin Guan, Xin‐Yuan Fu,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoRecent studies suggest that focal adhesion kinase (FAK) is important for cell migration. We now suggest a mechanism by which FAK activates the signal transducer and activator of transcription (STAT) pathway, regulating cell adhesion and migration. In particular, we observe that FAK is capable of activating Stat1, but not Stat3. Co-immunoprecipitation and in vitro binding assays demonstrate that Stat1 is transiently and directly associated with FAK during cell adhesion, and Stat1 is activated in this process. FAK with a C-terminal deletion (FAKΔC14) completely abolishes this interaction, indicating this association is dependent on the C-terminal domain of FAK, which is required for FAK localization at focal contacts. Moreover, Stat1 activation during cell adhesion is diminished in FAK-deficient cells, correlating with decreased migration in these cells. Finally, we show that depletion of Stat1 results in an enhancement of cell adhesion and a decrease in cell migration. Thus, our results have demonstrated, for the first time, a critical signaling pathway from integrin/FAK to Stat1 that reduces cell adhesion and promotes cell migration. Recent studies suggest that focal adhesion kinase (FAK) is important for cell migration. We now suggest a mechanism by which FAK activates the signal transducer and activator of transcription (STAT) pathway, regulating cell adhesion and migration. In particular, we observe that FAK is capable of activating Stat1, but not Stat3. Co-immunoprecipitation and in vitro binding assays demonstrate that Stat1 is transiently and directly associated with FAK during cell adhesion, and Stat1 is activated in this process. FAK with a C-terminal deletion (FAKΔC14) completely abolishes this interaction, indicating this association is dependent on the C-terminal domain of FAK, which is required for FAK localization at focal contacts. Moreover, Stat1 activation during cell adhesion is diminished in FAK-deficient cells, correlating with decreased migration in these cells. Finally, we show that depletion of Stat1 results in an enhancement of cell adhesion and a decrease in cell migration. Thus, our results have demonstrated, for the first time, a critical signaling pathway from integrin/FAK to Stat1 that reduces cell adhesion and promotes cell migration. extracellular matrix 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside glutathioneS-transferase Src homology epidermal growth factor focal adhesion kinase signal transducer and activator of transcription Janus kinase 1,4-piperazinediethanesulfonic acid hemagglutinin kinase-defective whole cell extract phosphate-buffered saline fibronectin interferon bovine serum albumin protein-tyrosine kinase Dulbecco's modified Eagle's medium fetal bovine serum electrophoretic mobility shift assay Extracellular matrix (ECM)1 proteins and integrins play essential roles in the regulation of cell adhesion and migration (1Gumbiner B.M. Cell. 1996; 84: 345-357Abstract Full Text Full Text PDF PubMed Scopus (2948) Google Scholar, 2Lauffenburger D.A. Horwitz A.F. 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Science. 1997; 277: 1630-1635Crossref PubMed Scopus (3401) Google Scholar) expression. In addition to JAK family kinases, which mediate signals from cytokine receptors, many kinds of protein-tyrosine kinases (PTKs) also activate STAT proteins under a variety of physiological or pathological conditions. In particular, EGF receptor kinase can directly activate STAT proteins (30Fu X.-Y. Zhang J.J. Cell. 1993; 74: 1135-1145Abstract Full Text PDF PubMed Scopus (273) Google Scholar, 31Ruff-Jamison S. Chen K. Cohen S. Science. 1993; 261: 1733-1736Crossref PubMed Scopus (241) Google Scholar, 32Sadowski H.B. Shuai K. Darnell Jr., J.E. Gilman M.Z. Science. 1993; 261: 1739-1744Crossref PubMed Scopus (642) Google Scholar, 33Quelle F.W. Thierfelder W. Witthuhn B.A. Tang B. Cohen S. Ihle J.N. J. Biol. Chem. 1995; 270: 20775-20780Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 34Leaman D.W. Pisharody S. Flickinger T.W. Commane M.A. Schlessinger J. Kerr I.M. Levy D.E. Stark G.R. Mol. Cell. Biol. 1996; 16: 369-375Crossref PubMed Scopus (206) Google Scholar). Interestingly, STAT activation by EGF results in inhibition of cell proliferation and apoptosis, which contrasts with the well documented EGF function of stimulation in cell growth, suggesting the STAT signaling pathway can negatively control cell growth (35Chin Y.E. Kitagawa M. Su W.-C.S. You Z.-H. Iwamoto Y. Fu X.-Y. Science. 1996; 272: 719-722Crossref PubMed Scopus (732) Google Scholar, 36Chin Y.E. Kitagawa M. Kuida K. Flavell R.A. Fu X.Y. Mol. Cell. Biol. 1997; 17: 5328-5337Crossref PubMed Scopus (471) Google Scholar). Furthermore, fibroblast growth factor receptor kinase, and Src family kinases may also activate STAT proteins (37Yu C.L. Meyer D.J. Campbell G.S. Larner A.C. Carter-Su C. Schwartz J. Jove R. Science. 1995; 269: 81-83Crossref PubMed Scopus (824) Google Scholar, 38Cao X.M. Tay A. Guy G.R. Tan Y.H. Mol. Cell. Biol. 1996; 16: 1595-1603Crossref PubMed Scopus (341) Google Scholar, 39Su W.C. 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Furthermore, we show that Stat1 activation reduces cell adhesion and stimulates cell migration. 293T and A431 cells were cultured in DMEM with 10% or 5% fetal bovine serum (FBS, Life Technologies, Inc.). Stat1 −/− and wild type fibroblast cells were cultured in RPMI 1640 with 10% FBS. U3A was cultured in DMEM with 10% FBS and 250 μg/ml hygromycin B. U3A-pSG5 and U3A-Stat1 cells were cultured in DMEM with 10% FBS, 250 μg/ml hygromycin B (Roche Molecular Biochemicals) and 500 μg/ml Geneticin (Life Technologies, Inc.). FAK wild type and −/− fibroblasts were cultured in DMEM (high glucose) with 10% FBS, 1 mm sodium pyruvate (Life Technologies, Inc.), 100 μm non-essential amino acids (Life Technologies, Inc.), and 100 μm 2-mercaptoethanol (Sigma). Expression vectors encoding HA-tagged wild type Stat1 and Stat1-SH2RQ mutant were described previously (30Fu X.-Y. Zhang J.J. Cell. 1993; 74: 1135-1145Abstract Full Text PDF PubMed Scopus (273) Google Scholar). Stat1-CYF has a single amino acid change on position 701 from Tyr to Phe. Plasmid pCX-Stat1 was constructed by inserting non-tagged Stat1 into StuI site of pCX vector that has a cytomegalovirus promoter. Expression plasmids encoding HA-tagged wild type FAK, kinase-defective mutant FAK (KD), Y397F mutant, and C-terminal 14-amino acid deletion mutant FAK ΔC14, had been described previously (42Chen H.C. Guan J.L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10148-10152Crossref PubMed Scopus (478) Google Scholar, 43Chen H.C. Appeddu P.A. Parsons J.T. Hildebrand J.D. Schaller M.D. Guan J.L. J. Biol. Chem. 1995; 270: 16995-16999Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar, 44Zhao J.H. Reiske H. Guan J.L. J. Cell Biol. 1998; 143: 1997-2008Crossref PubMed Scopus (302) Google Scholar). Rabbit polyclonal anti-Stat1 antibody (C-24) and monoclonal anti-Stat1 antibody (C-111) (Santa Cruz Biotechnology) were used for immunoprecipitation, Western blotting, supershift assay, and immunofluorescence staining. A monoclonal anti-FAK antibody (Transduction Laboratories and PharMingen) was used for immunofluorescence staining. Another rabbit polyclonal anti-FAK antibody (C-20) (Santa Cruz Biotechnology) was used for immunoprecipitation, Western blotting, and immunofluorescence staining. A rabbit polyclonal anti-phospho-Stat1 antibody (New England Biolabs) was used for immunoblotting; mouse anti-HA antibody (12CA5) and a rabbit polyclonal anti-FAK antibody (described previously (see Refs. 42Chen H.C. Guan J.L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10148-10152Crossref PubMed Scopus (478) Google Scholarand 43Chen H.C. Appeddu P.A. Parsons J.T. Hildebrand J.D. Schaller M.D. Guan J.L. J. Biol. Chem. 1995; 270: 16995-16999Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar)) were used for immunoprecipitation, Western blotting, and immunofluorescence staining. Polyclonal anti-Stat2 (45Fu X.Y. Kessler D.S. Veals S.A. Levy D.E. Darnell Jr., J.E. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8555-8559Crossref PubMed Scopus (343) Google Scholar) and anti-NF-κB/p65 (C-20) (Santa Cruz Biotechnology, Inc.) antibodies were used for in vitro assays. A monoclonal anti-vinculin antibody was used for immunofluorescence staining (Upstate Biotechnology Inc.). Tissue culture plates were coated overnight with 10 μg/ml human plasma fibronectin (Life Technologies, Inc.) in 1× PBS, washed twice with PBS, and then incubated with 2 mg/ml heat-inactivated (1 h at 70 °C) BSA in 1× PBS for 2 h at 37 °C. Cells were harvested by brief trypsinization and washed twice with PBS containing 0.5 mg/ml soybean trypsin inhibitor (Sigma). The cells were resuspended in DMEM without serum and added to coated plates (100 mm) at 8 × 106. After various times of incubation at 37 °C, cells were washed twice with cold PBS and lysed in whole cell-extract (WCE) buffer (15 mm Hepes, pH 7.9, 250 mm NaCl, 0.5% Nonidet P-40, 10% glycerol, and 1 mm EDTA) containing a mixture of protease and phosphatase inhibitors (0.5 mmphenylmethylsulfonyl fluoride, 1 mg/ml leupeptin, 1 mg/ml aprotinin, 1 mg/ml pepstatin, 1 mm vanadate, 10 mm NaF, and 1 mm dithiothreitol), left on ice for 45 min, and centrifuged for 10 min at 4 °C. WCE containing the same amount of total proteins were subjected to EMSA with 10 fmol of32P-labeled high affinity SIE probe (5′-AGCTTCATTTCCCGTAAATCCCTAAAGCT-3′) as described previously (35Chin Y.E. Kitagawa M. Su W.-C.S. You Z.-H. Iwamoto Y. Fu X.-Y. Science. 1996; 272: 719-722Crossref PubMed Scopus (732) Google Scholar). Forty-eight hours after transfection, cells were fixed by 1% glutaraldehyde (in PBS) in 37 °C for 15 min. Cells were stained with 0.2% X-gal (Amersham Pharmacia Biotech) (in 10 mm Na3PO4, pH 7.0, 150 mm NaCl, 1 mm MgCl2, 3.3 mm K4Fe(CN)6, 3.3 mmK3Fe(CN)6) for 1 h, washed with 70% ethanol, then covered with PBS. The number of blue-stained and transfected cells was counted in three different fields under microscopy (magnification, ×100). All experiments were repeated at least three times. For immunoprecipitation, cells plated on fibronectin were lysed with WCE buffer. Four hundred micrograms of proteins were incubated with anti-HA, anti-FAK (C-20), or anti-Stat1 (C-24) antibodies at 4 °C overnight. Twenty-five microliters of protein G- or protein A-agarose was added for 3 h of additional incubation at 4 °C. After washing the precipitates three times with WCE buffer with protease and phosphatase inhibitors, samples were electrophoresed in 6% or 8% SDS-polyacrylamide gels. Following electrophoresis, proteins were transferred to polyvinylidene difluoride membrane and blotted with anti-HA, C-24 anti-Stat1, C-20 anti-FAK, or anti-phospho-Stat1 antibodies. For immunoblot, 10 μg of proteins from each sample were analyzed. The cDNAs of Stat1 (pSG-Stat1) and FAK (pBluescript-FAK) were in vitrotranscribed and translated using the TNT Coupled Reticulocyte Lysate systems or TNT Coupled Wheat Germ Lysate systems (Promega) in the presence of Redivue l-[35S]methionine (>1,000 Ci/mmol at 10 mCi/ml; Amersham Pharmacia Biotech). Stat1 protein from in vitro translation reaction was mixed in a kinase reaction buffer (10 mm PIPES, pH 7.0, 5 mm MnCl2, 1 mm NaCl, 0.1 mm dithiothreitol, and 10 μm ATP) (38Cao X.M. Tay A. Guy G.R. Tan Y.H. Mol. Cell. Biol. 1996; 16: 1595-1603Crossref PubMed Scopus (341) Google Scholar) with insect cell SF21 lysates with or without FAK expression. After a 20-min incubation period at 30 °C, the samples were immunoprecipitated with an anti-Stat1 antibody (C-24) or non-related serum at 4 °C and applied to SDS-polyacrylamide gel electrophoresis as described above. Similar procedures were applied for other in vitrotranslation reactions. Stat1 protein levels were detected by direct autoradiography. Immunoprecipitation was performed in the buffer containing 15 mm Hepes, pH 7.9, 400 mm NaCl, 0.5% Nonidet P-40, 10% glycerol, and 1 mm EDTA. C-24 anti-Stat1, C-20 anti-FAK, C-20 anti-NF-κB/p65 (all from Santa Cruz Biotechnology, Inc.), and anti-Stat2 antibodies (45Fu X.Y. Kessler D.S. Veals S.A. Levy D.E. Darnell Jr., J.E. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8555-8559Crossref PubMed Scopus (343) Google Scholar) were used. Non-related serum was used as a control. The GST-Stat1 construct was generated by inserting a full-length Stat1 cDNA fragment from pSG-Stat1 (released byEcoRI) into pGEX-4T-3 (Amersham Pharmacia Biotech)EcoRI site. The GST-Stat1 and GST proteins were produced and purified according to the manufacturer's instructions. In vitro translated FAK was pre-cleared by GST-conjugated glutathione-Sepharose 4B beads and then incubated with 10 μg of purified GST-Stat1, or GST-conjugated glutathione-Sepharose 4B beads in the WCE buffer for 4 h at 4 °C. Following three washes with WCE buffer, the precipitates were separated by SDS-polyacrylamide gel electrophoresis and visualized by autoradiography. Cells were collected and treated (as described under "Cell Extracts and EMSA") before plating in six-well plates on fibronectin-coated coverslips. After incubation for various periods of time, cells were washed with PBS twice, and fixed with 4% paraformaldehyde for 30 min, permeabilized with 0.5% Triton X-100 for 5 min, blocked with 3% BSA for 1 h, and then processed for immunofluorescence by using different primary antibodies. IFN-γ-treated cells were also cultured on coverslips (no fibronectin) overnight before treatment. Thirty minutes after treatment, cells were fixed as above. The primary antibodies were diluted as follows: α-HA, 1:100; monoclonal α-FAK and α-Stat1 (C-111), 1:100; polyclonal α-FAK and α-Stat1 (C-24), 1:300. The secondary antibodies were fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (Alexa™ 488, Molecular Probes, 1:200) or Texas Red-conjugated goat anti-mouse IgG (Jackson Immunoresearch Laboratories, Inc., 1:200). Different concentrations of human plasma fibronectin (FN) (Life Technologies, Inc.) were adsorbed onto plastic 96-well tissue culture plates (100 μl/well). After using 0.5% BSA to block the plates in 37 °C, certain numbers of cells depending the cell types (see figure legends), were plated and incubated at 37 °C to indicating time points. The plates were washed with PBS twice, and cells were fixed with 4% paraformaldehyde, pH 7.4, for 30 min in 4 °C. Cells were washed again, stained with 0.5% crystal violet, and incubated overnight at room temperature. The extent of cell adherence was determined by plate reader at OD630. Migration assays in 24-well transwell chambers (8-μm pore size, Costar) were carried out as described previously (46Cary L.A. Chang J.F. Guan J.L. J. Cell Sci. 1996; 109: 1787-1794Crossref PubMed Google Scholar). Briefly, 0.6 ml of serum-free medium with 10 μg/ml fibronectin was added to the lower chamber, whereas cells were added into the upper chamber in serum-free medium. After an indicated time of incubation at 37 °C to allow cells to migrate, membranes were fixed with 3% paraformaldehyde, pH 7.4, for 30 min in 4 °C and stained. Cells that did not migrate were removed by wiping the upper side of membranes, and the migrated cells were counted under a microscope (magnification, ×100). Six different views were counted. We first determined whether FAK could induce Stat1 activation. In this experiment, 293T cells were transfected with vectors expressing FAK and Stat1 separately, or in combination. Using a gel EMSA, we observed Stat1 activation in cells transfected with FAK (Fig. 1 A, lane 4) but not in mock-transfected cells (Fig. 1 A,lane 2), suggesting that FAK activated endogenous Stat1 in vivo in these cells. This result was confirmed by a supershift assay (data not shown). Transfection of a HA-tagged Stat1 (30Fu X.-Y. Zhang J.J. Cell. 1993; 74: 1135-1145Abstract Full Text PDF PubMed Scopus (273) Google Scholar) also generated a weak Stat1 complex, which migrated slightly slower than the endogenous Stat1 complex, possibly due to the added HA tag in the protein (Fig. 1 A, lane 3). More impressively, in cells co-transfected with FAK and Stat1, Stat1 was strongly activated (Fig. 1 A, lane 5). This Stat1 complex was recognized by an anti-Stat1 antibody, forming a supershifted complex (SS) in the EMSA (Fig. 1 A, lane 6). To investigate whether this Stat1 activation by FAK-cotransfection is specific for Stat1, we further assessed possible activation of Stat3 by FAK under the same conditions. In contrast to Stat1, Stat3 was weakly activated when Stat3 and FAK were co-transfected (Fig. 1 B). However, Src could activate Stat1 as well as Stat3 at similar levels in co-transfection studies. These results indicate that Stat1, not Stat3, is a preferable target of FAK signaling. More importantly, endogenous Stat1, not Stat3 or other STAT proteins, appeared to be activated in cells transfected with FAK only, although there are several members of endogenous STAT proteins in these cells (Fig. 1 A,lane 4). Jak1 kinase is required for tyrosine phosphorylation of Stat1 in response to many cytokines (34Leaman D.W. Pisharody S. Flickinger T.W. Commane M.A. Schlessinger J. Kerr I.M. Levy D.E. Stark G.R. Mol. Cell. Biol. 1996; 16: 369-375Crossref PubMed Scopus (206) Google Scholar). However, Jak1 was not necessary for the STAT activation by FAK, since Stat1 was activated by FAK in a Jak1-deficient HeLa cell line, E2A4 (47Loh J.E. Schindler C. Ziemiecki A. Harpur A.G. Wilks A.F. Flavell R.A. Mol. Cell. Biol. 1994; 14: 2170-2179Crossref PubMed Google Scholar), similarly to results observed in 293T cells (Fig. 1 B,lane 10). To visualize the effect of Stat1 activation on cell adhesion, these cells were co-transfected with a vector that expressed β-galactosidase. Thus, transfectants could be specifically recognized by the blue color after X-gal staining. We observed dramatic morphological changes in transfected cells that seemed to parallel Stat1 activation by FAK. The FAK- and Stat1-co-transfected cells clearly lost their cell spreading ability and were detached from the plate (Fig. 1 C, e; arrows indicatelight blue cells, which were transfected at a lower level.). For the cells transfected with FAK alone, a portion of transfected cells also underwent similar morphological alterations that might result from the endogenous Stat1 activity induced by FAK (Fig.1 C, d). The cells that were mock-transfected or Stat1 only transfected showed no effect (Fig. 1 C,a and b). In contrast to cells co-transfected with FAK and Stat1, those co-transfected with FAK and Stat3 showed no significant change on morphology (Fig. 1 C, f). Therefore, these data further demonstrate that Stat1 activation by FAK, but not Stat3, can negatively affect cell adhesion. We next determined the functional domains that were involved in Stat1 activation by FAK. The C-terminal tyrosine (Stat1-Y701) or the SH2 domain of Stat1 is essential for Stat1 activation in response to cytokines. We found that mutations of the critical C-terminal tyrosine (Stat1-CYF) or of the SH2 domain (Stat1-SH2RQ) also prevented Stat1 activation by FAK (Fig.2 A, compare lanes 6 and 8 with lane 4). Almost equal levels of Stat1 protein in the various transfected cells were verified by a Western blot analysis (Fig. 2 A,lower panel). Similarly, expression of wild type Stat1 in these cells occasionally resulted in a low level of Stat1 activation (Fig. 2 A, lane 2); however, expression of either C-terminal tyrosine mutant (Stat1-CYF) or SH2 mutant (Stat1-SH2RQ) alone did not generate this Stat1 activation (Fig.2 A, lanes 5 and 7). A weak Stat1 activity was observed in cells co-expressing either of these two mutant Stat1 proteins with FAK. This might be attributed to endogenous STAT activation by FAK, as was also observed in cells transfected by FAK alone (Fig. 2 A, lane 3). These results indicate that the C-terminal tyrosine and the SH2 domain are essential for Stat1 activation by FAK. To verify whether the kinase activity of FAK is required for the STAT activation, we used kinase-defective FAK with a K454R mutation at the ATP binding site of the catalytic domain (48Hildebrand J.D. Schaller M.D. Parsons J.T. J. Cell Biol. 1993; 123: 993-1005Crossref PubMed Scopus (359) Google Scholar). This mutation of FAK (KD) dramatically reduced Stat1 activation compared with that by the wild type FAK (Fig. 2 B, compare lanes 5 and 6 with lanes 3 and4). The observed Stat1 activity (lane 6) was at the same level as that of Stat1 alone (lane 2), demonstrating that this FAK mutant was defective in Stat1 activation. Tyrosine 397 of FAK is a major autophosphorylation site of the protein and is required for the binding of Src family kinases. The Src-FAK association appeared to increase the tyrosine phosphorylation of FAK and other substrates (13Schaller M.D. Hildebrand J.D. Shannon J.D. Fox J.W. Vines R.R. Parsons J.T. Mol. Cell. Biol. 1994; 14: 1680-1688Crossref PubMed Scopus (1121) Google Scholar, 49Calalb M.B. Polte T.R. Hanks S.K. Mol. Cell. Biol. 1995; 15: 954-963Crossref PubMed Google Scholar). We found that this Src association site of Tyr-397 was also involved in the STAT activation, because a point mutation that replaced tyrosine 397 with phenylalanine (Y397F) significantly decreased Stat1 activation (Fig.2 B, lanes 7 and 8). These results suggest that the FAK is essential for Stat1 activation, and the Src binding may also be involved in further activation of Stat1. In the above experiments, the mutant FAK proteins were expressed at a level comparable to that of wild type FAK, whereas endogenous FAK expression in these cells was relatively low (see lower panel). After cytokine stimulation, STAT proteins can bind directly to phosphorylated receptor-tyrosine kinase complexes. Since the SH2 domain of Stat1 is required for Stat1 activation by FAK, we examined possible interaction between Stat1 and FAK. To avoid potential artifacts arising from protein overexpression, we performed the experiment using untransfected cells. An antibody specific to FAK was used to perform immunoprecipitation in untransfected 293T cells, followed by an examination of the immunoprecipitated complexes using an anti-Stat1 antibody (Fig. 3 A). In this assay, Stat1 was clearly co-immunoprecipitated with an anti-FAK antibody (lanes 1–4, upper panel). However, the migration of co-immunoprecipitated Stat1 was slower than that of the major Stat1 band (indicated asStat1), immunoprecipitated by an anti-Stat1 antibody (lane 5). We suspected that these slower migrating Stat1 bands were resulted from protein phosphorylation after Stat1 protein had interacted with FAK. This notion was confirmed by a protein blot with another antibody that specifically recognizes tyrosine-phosphorylated, but not unphosphorylated, Stat1. Only these slower migrating bands were recognized by this anti-phosphotyrosine Stat1 (Stat1p) antibody (Fig. 3 A, middle panel), whereas the major unphosphorylated Stat1 band (shown in lane 5, upper panel) was not recognized by this antibody (lane 5,middle panel). Intriguingly, it appeared that only tyrosine-phosphorylated Stat1 was co-immunoprecipitated with FAK, and this FAK-Stat1 association transiently reached the maximal level when cells were attached to fibronectin for a brief period (at 0.5-h time point). With the progression of cell attachment, the amount of Stat1 associated with FAK was significantly reduced. The levels of precipitated FAK protein were almost the same (Fig. 3 A,lower panel). These results suggest that Stat1 can associate transiently with FAK at early time points of cell adhesion when FAK is activated (7Guan J.L. Shalloway D. Nature. 1992; 358: 690-692Crossref PubMed Scopus (726) Google Scholar, 8Hanks S.K. Calalb M.B. Harper M.C. Patel S.K. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8487-8491Crossref PubMed Scopus (731) Google Scholar, 9Schaller M.D. Borgman C.A. Cobb B.S. Vines R.R. Reynolds A.B. Parsons J.T. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5192-5196Crossref PubMed Scopus (1295) Google Scholar). A similar observation was also made in A431 cells (Fig. 3 B) and U3A-Stat1 cells (data not shown) in which tyrosine phosphorylation of Stat1 was co-immunoprecipitated with FAK. Consistent with the transi
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